Allograft tissue purification process for cleaning bone

ABSTRACT

A method for producing a cleaned bone graft with osteoinductivity above 2.0 suitable for transplantation into a human. The first step is sonicating a bone graft in a nonionic detergent in an ultrasonic cleaner at a temperature ranging from about 33° C. to about 37° C. and for a time period ranging from 15 minutes to 2 hours effective to produce a cleaned bone graft essentially free from bone marrow. The bone graft is sonicated in purified water in an ultrasonic cleaner at a temperature ranging from about 33° C. to about 37° C. a plurality of times to remove the detergent producing a cleaned bone graft. The bone graft is then sonicated in hydrogen peroxide in an ultrasonic cleaner at a temperature ranging from about 33° C. to about 37° C. for a time period ranging from 10 minutes to about 2 hours effective to retain osteoinductivity of the bone graft and again sonicated in purified water in an ultrasonic cleaner at a temperature ranging from about 33° C. to about 37° C. a plurality of times to produce a cleaned bone graft. The final step is sonicating the bone graft in an alcohol at a temperature ranging from 33° C. to 37° C. for 30 minutes to 2 hours, all of the steps being effective to reduce any initially present viruses at least two logs and bacteria at least ten logs and the bone graft is finally sonicated in purified water to remove the alcohol.

RELATED APPLICATIONS

There are no related applications.

FIELD OF THE INVENTION

The present invention generally relates to a method for cleaning bonesto produce bone grafts suitable for transplantation into a human. Theprocess more specifically involves the use of ultrasonic cleaning in theremoval of bone marrow, tissue, bacteria and viruses by causingultrasonic cavitation in sequential detergent, hydrogen peroxide andalcohol solutions.

BACKGROUND OF THE INVENTION

One hundred to two hundred thousand tissue transplants are annuallyperformed in the United States. The Single most variable factor withrespect to allographic transplantation is the preparation of such boneand tissue segments. Procedure and protocol of the some 400 tissue banksin North America are quite varied and has resulted in various technologywith developed processes.

Allografts are vital for bone stock deficiencies that occur duringorthopaedic trauma, joint reconstruction, or other reconstructiveprocedures. The main criteria for an orthopaedic allograft are theretention of strength, the retention of biologic factors, and thereduction of risk of disease transmission. The first two should not beaffected by processing, while processing should eliminate the risk fordisease transmission.

There is no known industry standard specifying levels of cleanliness forcleaning and preparing bone segments. The problems associated with thislack of standards interpret to poor process control, inadequate removalof tissue from the parent surface and to a large extent lack ofsterility during the tissue recovery process.

Human bone obtained from cadaveric donors is typically procured understerile conditions in an operating suite environment of local hospitals.The bone is stored frozen until it is further processed into smallgrafts under similar sterile conditions, or under clean-room conditions.Procurement and processing of human tissues is typically performed bygroups certified by the American Association of Tissue Banks understandard operating procedures for the processing of each specific bonegraft. Large bones such as the femur are thawed and debrided of excesstissue prior to being cut into smaller grafts. Processing of the smallergrafts includes cleaning of bone marrow from the cancerous bone spaces.Cleaning of bone marrow and tissue from small bone grafts has beendescribed in the scientific literature and in brochures and documentsmade public by groups involved in the procurement and processing ofhuman tissues.

Osteotech, Inc. describes a bone graft cleaning process called Permein.TM. (“a combination of ethanol and non-ionic detergent” which involvesthe use of a solution of ethanol and detergent to clean bone grafts.

Detergents are amphophil compounds which facilitate solubilization ofrelatively insoluble lipids present in, for example, bone marrow, yet athigher concentrations tend to form micellar structures (Helenius, A. andSimons, K. Solubilization of Membranes by Detergents, Biochim. Biophys.Acta 415 (1975) 29-79). The formation of micellar structures tend tolimit the effective concentration range for detergent solutions and thussoaking of bone in a given volume of detergent solution may not betotally effective in that the absolute amount of detergent present islimited and if the amount of lipid material to be solubilized exceedsthe solubilization capability of the detergent present, lipidsolubilization will not be complete.

The use of prior art procedures to remove bone marrow involves the useof pressurized flow of solution as a rapidly moving stream whichdislodges bone marrow by impact of the solvent on the bone graft. Suchprocedures tend to generate aerosols of tissue and solvent which can behazardous to processing personnel. The present invention eliminates thishazard.

Ultrasonic cleaners are extensively used in cleaning glass tubes, metalinstruments, filers, etc. Ultrasound is sound transmitted at frequenciesbeyond the range of human hearing. Ultrasonic energy in liquid generatedby piezoelectric or other types of transducers creates cavitation, whichis the mechanism for ultrasonic cleaning. Cavitation consists of theformation and collapse of countless tiny cavities, or vacuum bubbles, inthe liquid. The energy produces alternating high and low pressure waveswithin the liquid of a tank. The liquid is compressed during the highpressure phase of the wave cycle, then pulled apart during the lowpressure phase. As the pressure in the liquid is reduced during the lowpressure phase, cavities grow from microscopic nuclei to a maximumcritical diameter. During the subsequent high pressure phase they arecompressed and implode. The energy is powerful, but safe for partsbecause it is localized at the microscopic, i.e., cellar, scale. Factorsaffecting the strength of cavitation are temperature, surface tension,detergents or other agents which reduce surface tension are optimal,viscosity (medium vapor pressure is most conducive to ultrasoundactivity), and density (where high density creates intense cavitationwith greater implosive force).

A number of prior art references have used ultrasonics together withdetergents and other solutions to clean bone.

In U.S. Pat. Nos. 5,556,379 issued Sep. 17, 1996 and 5,976,104 issuedNov. 2, 1999, it is noted that processing of the smaller grafts includescleaning of bone marrow from the cancerous bone spaces using mechanicalmeans, soaking, sonication, and/or lavage with pulsatile water flowunder pressure. This cleaning may use reduced or elevated temperatures,for example 4° C. to 65° C., and may also include the use of detergents,alcohol, organic solvents or similar solutes or combination of solutesdesigned to facilitate solubilization of the bone marrow.

In the Simonds reference from the New England Journal of Medicine, page726, Mar. 12, 1992, entitled TRANSMISSION OF HUMAN IMMUNODEFICIENCYVIRUS TYPE I FROM SERONEGATIVE ORGAN AND ISSUE DONOR, the bone waslyophilized and treated with ethanol. (Step 1 or 3). The lyophilizedtissue had soft tissue removed, followed by treatment with twoantibiotics (Step 2), irrigation with sterile water, packaging andrefreezing and lyophilization to a residual moisture content of lessthan 5%. The ethanol treated tissue underwent ultrasonic cleaning in 30%ethanol (Step 1), removal of marrow by water lavage and brief treatmentin 100% ethanol (Step 3).

Another pertinent reference is the article A Review of AllograftProcessing and Sterilization Techniques and Their Role in Transmissionof the Human Immunodeficiency Virus, American Journal of SportsMedicine, Vol 21, No. 2 ((1993) presented at the interim meeting ofAOSSM, February 1993, San Francisco Calif. (Exhibit 6) sponsored byLifeNet Transplant Services.

The article states in part: “Bone preserved by freeze-drying was firstthawed, and followed by removal of extraneous soft tissue. It was thenplaced in the ultrasonic cleanser in 30% ethanol solution. All marrowelements were removed by pulsating water lavage, followed by 15 minutesin 100% ethanol and brief irrigation with bacitracin and polymyxin Bsulfate solution. These tissues were then washed and packaged in glasscontainers and refrozen.”

U.S. Pat. No. 5,095,925 issued Mar. 17, 1992 is directed toward a bonecleaning device using ultrasonics which removes gross tissue from boneto prepare the same for transplant and use in surgery. The bone issubjected to a positive pressure stream of sterile water, ultrasonicallycleaning the same in a detergent followed by rinsing and soaking andreintroduced to the ultrasonic process if necessary within a preferredworking temperature range of 27° C. to 33° C.

U.S. Pat. No. 5,509,968 issued Apr. 26, 1996 is directed toward cleaningused orthopaedic implants which are decontaminated and made availablefor reuse by a three step process for removal of protein tissue, bonetissue and lipids.

The '968 patent uses a process to clean then implant in the followingmanner: The implant is suspended in an aqueous bath of detergentsuitable for emulsifying lipids at elevated temperatures, such as 40° C.to 60° C., and is typically treated for about 1 to 45 minutes by the useof an ultrasonic cleaning system. The solution in the treating corer isdiscarded and the container and implant are washed with clean water. Acontainer is filled with a dilute acid capable of dissolving bone salts(e.g., calcium phosphate minerals that are deposited in the collagenmatrix of the bone). The implant is added to the container, andsubjected to ultrasonic treatment for approximately the same time. Aftertreatment, the solution containing dissolved bone salts is discarded andthe implant and container are again rinsed with clean water. The implantis then subjected to a bath of an aqueous solution sodium hypochloriteof a concentration as sold for general cleaning purposes, (householdbleach). This step removes any remaining organic bone tissue as well asprotein. An ultrasonic cleaning system is again used for the same timeand temperature. When this step is completed, the solution is discardedand both the implant and container rinsed with water.

In U.S. Pat. No. 5,333,626 issued Aug. 2, 1994, a high pressure wash isused to clean bone. The bone is cleaned with a high pressure detergentsolution such as TritonX-100 and Tween 80 preferably from 37° C. to 80°C. Following the washing, the solution is changed. Sterile water orbiologically acceptable alcohol is used to remove the detergent and itis removed by rinsing with sterile water. The bone may be furtherdecontaminated by exposing it to 3% hydrogen peroxide solution from 5 to120 minutes (preferably 5 to 60 minutes) after which the residualhydrogen peroxide is removed by washing with sterile water. Aftercleaning, the bone is finally decontaminated by contacting the bone witha global decontaminate for 30 to 60 minutes. U.S. Pat. No. 5,797,871issued Aug. 25, 1998 is also directed toward a bone cleaning processusing ultrasonics in which the bone is sonicated in a solution ofseveral detergents within a temperature range of 37° C. to 50° C. toproduce bone grafts essentially free from bone marrow and detectablefungal and viral contamination.

SUMMARY OF THE INVENTION

The present invention is directed toward a process for cleaning bones toremove tissue, bone marrow, bacteria, fungi and viruses by sonicatingthe bone in a nonionic detergent solution, draining the detergentsolution and respectively soaking and sonicating the bone in a purifiedwater bath to remove the detergent, draining the water bath and adding ahydrogen peroxide solution in which the bone is sonicated. The hydrogenperoxide is drained and the bone is soaked and sonicated in purifiedwater to remove hydrogen peroxide and soaking and sonicating the bone inan alcohol solution, draining the same and soaking and sonicating thebone in purified water to remove the alcohol. The process arrives at abone which has a viral clearance of at east two logs and retains itsosteoinductivity.

An object of the present invention is to provide a means of removingbone marrow from the luminal and cancellous bone spaces in essentiallyintact bone grafts and small machined, shaped bone grafts.

It is a further object of the invention to process essentially intactbone grafts with minimal residual bone marrow as bone marrow may harborpotential viral particles and/or viral genomes integrated into thegenomes of specific cell types present in the bone marrow, thus reducingthe potential for transmission of infective agents such as bacteria andviruses.

Another object of this invention is to use detergent and hydrogenperoxide solutions in the processing of bone grafts. Hydrogen peroxideand detergents have been demonstrated to be virucidal towards virusessuch as HIV and hepatitis and certain bacteria, and to enhancecavitation associated with ultrasonic cleaners. Alcohol and detergentsolutions also offer advantages of enhancing solubilization of bonemarrow, reducing surface tension properties of aqueous solutions, andinactivating viruses and bacteria.

Still another object is to use ultrasonic cleaners which offeradvantages of cavitation events which facilitate disruption andbreakdown of soft tissues at the microscopic level.

It is yet another object of the invention that the present invention hasparticular utilization in the medical field where the article beingcleaned is a surgical device for allograft, autograft, xenograft andartificial transplants.

It is a particular object of this invention to provide a system for thesterile preparation of transplantable tissue. The needs filled by saidsystem on a broad scale will be a unification of procedure and protocolfor allograft tissue preparation, consistent performance and results oftissue processing centers, reduced detrimental effects of toxicchemicals and radiation now used by several processors in the bonebanking community, increase in the quality of allograft materialsproduced, decreased post-operative infection and transmission ofdisease, negation of local environmental factors such as toxic andpollutants and quicker functional incorporation of transplantedallografts.

It is another object of the present invention to provide a system forthe sterile preparation of transplantable tissue that will avoid theapplication of secondary sterilants that produce deleterious effects inthe host site and body.

It is yet another object of the present invention to provide a systemfor the sterile preparation of transplantable tissue that will increasethe shelf life, product quality, and component integrity, deriveconsistent and uniform results of prepared tissue and reduce thepreparatory expense.

These and other objects, advantages, and novel features of the presentinvention will become apparent when considered with the teachingscontained in the detailed disclosure which along with the accompanyingdrawings constitute a part of this specification and illustrateembodiments of the invention which together with the description serveto explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart diagram of a preferred embodiment of the presentmethod for producing a cleaned bone graft; and

FIG. 2 illustrates a flow chart diagram of an alternative embodiment ofthe present method for producing a cleaned bone graft;

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions.

The below definitions serve to provide a clear and consistent understandof the specifications and claims, including the scope to be given suchterms.

Bone Graft. The term “Bone Graft” is intended any bone obtained from acadaver donor, for example any shaped bone part and/or any small cutpieces of bone.

Cleaning Container. By the term “cleaning container” is intended for thepurpose of the present invention any container of a size sufficient tocontain the bone graft being processed. The cleaning container used wasa 4 liter stainless steel container with wire mesh to support the bonegraft.

Detergent. By the term “detergent” is intended any agent which through asurface action that depends on it possessing both hydrophilic andhydrophobic properties and/or exerts oil-dissolving (cleansing) and/orantibacterial and/or antiviral effects.

Ultrasonic Cleaner. The term “ultrasonic cleaner” is intended anyultrasonic cleaning device capable of operating from 20 kHz to 1000 kHz,preferably at about 40 kHz with an energy level ranging from 10-180W/sq.in., and includes, for example, but not by way of limitation, aBranson 8000 Ultrasonic Cleaner Model Numbers: 84540-18 or any similarultrasonic cleaner.

Bone Marrow. The term “bone marrow” is intended for the purposes of thepresent invention the highly cellular hematopoietic connective tissuefilling the medullary cavities and spongy epiphyses of bones which mayharbor bacterial and/or viral particles and/or fungal particles.

Solvent. The term “solvent” is intended for the purposes of the presentinvention, a liquid cleaning composition capable of: facilitating thesolubilization of lipid, facilitating bone marrow removal, inactivatingviral and/or bacterial particles, and/or disrupting cell membranes,which may contain, but is not limited to, one or more of the following:sterile water; saline; a detergent; an alcohol, for example, ethanoland/or isopropanol or a combination of same, solvents, a combination ofsolutes desired to facilitate solubilization of bone marrow, forexample, chelating agent; virucidal agent; bacteriocidal agent;antimycotic agent; sodium hydroxide or similar strong base, organicand/or inorganic acid and hydrogen peroxide.

While the present invention and best mode of the invention is shown inFIG. 1 and will be described in connection with certain preferredembodiments, it is not intended that the present invention be solimited. On the contrary, it is intended to cover all alternatives,modifications, and equivalent arrangements as may be included within thespirit and scope of the invention as defined by the appended claims.

The present invention is directed toward the cleaning and processing ofbone grafts using a nonionic detergent soak (see Table 1), a hydrogenperoxide soak, and 70% alcohol (ethanol and isopropanol) soak, andfrequent intermittent purified water washes, all under temperaturecontrolled sonication. In the process, bone graft samples were processedas control (0-hour H₂O₂ treatment, with no sonication) or treatment(5-hour H₂O₂ treatment). Hydrogen peroxide (H₂O₂) is an oxidizingchemical used to process bone allografts with the potential to eradicatemicroorganisms and viruses. It was previously thought that hydrogenperoxide could potentially compromise osteoinductivity and bonestructural proteins.

Compression cylinders (5.3-mm×5.3-mm) of the bone grafts were fabricatedfrom human femurs (age 39M & 61F) oriented longitudinally andtransversely, and were preserved both frozen (−70° C.) and freeze-dried(N=8 for all groups). Freeze dried samples were rehydrated for at least1-hour prior to testing and frozen samples were soaked for at least15-minutes prior to testing, both in normal saline. Samples were loadedto failure in uniaxial compression at a strain rate of 0.01 per secondand maximum and yield stress were calculated. Impact specimens werefabricated into anterior cervical fusion (ACF) allografts from fibulas(age 46M, 21M, 60M, & 62M), and were preserved both frozen andfreeze-dried (N=5 for all groups). Samples were secured into a customfixture using 3 N-m of torque and impacted starting at 5-cm with 1-cmincrements, using an ACF impact tool and an 841-g carriage, untilfailure. Total kinetic energy at failure was calculated for each ACF.

Osteoinductivity: Cortical bone from three donors (37M, 49M, 58F) wasprocessed using a control process, 0 hour H₂O₂ with no sonication, atreatment process with 1 hour, 3 hours, and 5 hours of H₂O₂ treatment,and a negative control. After processing, the bone was ground,demineralized, and prepared into 32% Demineralized Bone Matrix in ahyaluronan carrier. Samples (15 mg) were implanted bilaterally into thehamstring muscle in an athymic mouse model, approved by the Universityof Medicine and Dentistry of New Jersey animal care and use committee.Implants were evaluated histologically after 28 days.

Viral Clearance: Cortical bone samples were processed with a 1-hour H₂O₂step. Samples were subjected to the previous steps of the process. Foreach of six representative viruses, for each step of the process,samples were spiked with a virus suspension, and subjected to the giventreatment step, while a control was subjected to an inert, zero-time,but equal-volume version of the same step. Supernatant was recoveredfrom these samples, neutralized (where appropriate), and assayed forviral activity, using plaque and similar assays. Viral reduction foreach virus for each step was calculated as the difference between theviral titer of the control, and the viral titer of the test sample atthe full cycle time for the given treatment step. Results are the sum ofthe log reductions for all treatment steps for each virus.

Bacteriological Sterility: Cortical bone samples were subjected to theprevious steps of the process. For each of six representative bacteria,for each step of the process, sample surfaces were treated at designatedprotocol bacteria concentration and subjected to a one quarter timeincrement of the preferred treatment step and a fill time increment ofthe pre treatment, while a control was subjected to an inert, zero-time,but equal-volume version of preferred treatment step. The samples wereassayed for bacteria activity, using standard assays. Bacteria reductionfor each of the bacteria was calculated as the difference between theorganism titer of the control and the organism titer of the test sampleat one quarter cycle time (t₁ Chart 2) and the fill cycle time (t₂ Chart2) for the given treatment step. Results are the sum of the logreductions for all treatment steps for each bacteria.

Results in Testing

Mechanical: The results of the compression testing show no significantdifferences between the control and treatment group maximum stress data(Table 2). Results of the impact testing revealed no significantdifferences between the control and treatment groups (Table not shown;the means (standard deviation) are: control=49.8 (45.7); treatment=35.2(22.6)).

Osteoinductivity: Hydrogen peroxide cleaning had a statisticallysignificant effect on osteoinductivity, giving a linear decrease withnag peroxide time (Chart 1). The mean (SD) osteoinductivity scores were3.65 (0.49) for 0 hours, 3.04 (0.97) for 1 hour, 2.57 (1.36) for 3hours, 1.47 (1.10) for 5 hours H₂O₂ treatment times. The negativecontrol score was zero. Compared to the control (0 h), the 1 hour scorewas not significantly different (p=0.113), and the 3 hour and the 5 hourscores were significantly different (p=0.045 & p=0.0001, respectively).

Under current osteoinductivity stands, a score of 4.0 to 3.0 is highlyosteoinductive, 3.0 to 2.0 is moderately osteoinductive, 2.0 to 1.0 isslight osteoinductive.

Viral Clearance: The results of the viral clearance study demonstratethat processing the cortical bone allografts in a nonionic detergent,H₂O₂, and alcohol gives viral reductions greater than six logs in allcases except the PPV virus (Table 3). Under FDA definitions, viruses canbe listed as cleared at two logs or better. It should be specificallynoted that in the present invention, HIV virus is reduced onequadrillion times (10¹⁵).

Processing the bone graft with a 5-hour H₂O₂ soak does not affect thecompression strength of cortical bone allografts. Likewise, the impactdata did not show any statistical differences. The osteoinductivityscore for the 1-hour H₂O₂ treatment time is favorable, because nosignificant statistical decrease was seen. The 3 hours and 5 hourstreatment times were undesirable, as they caused statisticallysignificant decreases in osteoinductivity. The viral clearance resultverifies that the risk for disease transmission can be greatly reducedor eliminated by processing, beyond standard donor testing and screeningprocedures.

Bacteriological Sterility: The results of the bacteriological sterilitystudy demonstrate that processing the cortical bone allografts in anonionic detergent, H₂O₂, and alcohol soaks gives bacteria reductionsgreater than ten logs. Under FDA definitions, bacteriological reductionfor a single step is considered effective at two logs or better. Itshould be noted that one bacteria Clostridium Sporogenes was not testedunder an acceptable protocol and thus is not listed in Chart 2 below.

The bacteria Candida albicans, Staphylococcus aureus, Staphylococcusepidermidis, Escherichia coli, Pseudomonas aeruginosa, and Bacillussubtilis were all reduced after complete treatment at least ten logs.The bacteriological sterility result verifies that the risk for diseasetransmission from bacteria can be greatly reduced or eliminated byprocessing, beyond standard donor testing and screening procedures.

CHART 2 Microbiological Reduction Results Microbiological Log ReductionsOrganism Process Step T₁ T₂ T₁ Total T₂ Total Candida albicans TritonX-100 1.78 1.58 >11.11 >10.74 H₂O₂ >5.47 >5.10 Alcohol >5.64 >5.64Staphylococcus aureus Triton X-100 −0.59 0.20 >11.78 >11.78H₂O₂ >5.93 >5.93 Alcohol >5.85 5.85 Staphylococcus Triton X-100 0.560.41 >10.82 >10.82 epidermidis H₂O₂ >5.21 >5.21 Alcohol >5.61 >5.61Escherichia coli Triton X-100 0.03 1.82 >10.44 >10.44 H₂O₂ >5.19 >5.19Alcohol >5.25 >5.25 Pseudomonas aeruginosa Triton X-100 2.483.40 >13.70 >13.70 H₂O₂ >5.09 >5.09 Alcohol >5.21 >5.21 Bacillussubtilis Triton X-100 2.56 2.41 >11.11 >11.28 (vegetative)H₂O₂ >5.25 >5.25 Alcohol 3.30 3.62

Overall, these results demonstrate that it is possible to clean corticalbone allografts without causing a reduction in mechanical strength or asignificant loss in osteoinductivity, while at the same timesignificantly reducing the risks of disease transmission.

TABLE 1 List of Non-ionic Detergents N,N-Dimethyldodecylamino-N-oxideOctylglucoside Polyoxyethylene (PEG) alcoholsPolyoxyethylene-p-t-octylphenol Polyoxyethylene nonylphenolPolyoxyethylene sorbitol esters Polyoxy-propylene-polyoxyethylene estersp-isoOctylpolyoxy-ethylene-phenol formaldehyde polymer

TABLE 2 Compression maximum stress data (MPa) comparing the controlgroups to the 5-hour H₂O₂ treatment test groups Tissue OrientationLongitudinal Transverse Storage Donor Info Control Test Pr (F) ControlTest Pr (F) Frozen 39m 164 (7)  159 (9)  0.25 128 (9)  119 (10) 0.07 61f156 (5)  159 (8)  0.42 124 (11) 121 (9)  0.52 Freeze-Dried 39m 219 (27)222 (27) 0.82 153 (20) 167 (25) 0.24 61f 206 (27) 202 (38) 0.82 127 (15)117 (15) 0.21 Means are presented with their corresponding standarddeviations in parentheses. For all groups, N = 8. The probability thatthe means are equal is given as Pr (F). Values < 0.05 are consideredstatistically different.

TABLE 3 Total viral clearance in cortical bone allograft due toprocessing in detergent, 1-hour hydrogen peroxide, and alcohol Total LogReduction of Virus RNA or DNA Model for Virus Enveloped Viruses BVDV(bovine viral RNA Hepatitis C >10.62 diarrhea) (HCV) HIV (human RNAHIV >15.22 immunodeficiency) PrV (Pseudorabies) DNA CMV/Herpes >12.23Non-Enveloped Viruses HAV (Hepatitis A) RNA HAV >6.46 Polio RNAPolio/pico- >10.96 rnaviridae PPV (porcine parvovirus) DNA Human 2.57parvovirus B19

Process 1:

In the allograft tissue purification process, cortical bones are takenfrom the normal bone recovery process where they have been frozen andshipped or stored for processing. The bone tissue was thawed in aGentamicin soak (3.1 g Gentamicin in 4000 ml water) for 15 minutes to 2hours. A debridement process was performed, gross cleaning the bonesample with a wire wheel or scalpel for 30 minutes. Condyles were cutfrom the long bones and cortical and cancellous tissue was separated.Processing was begun on the cortical tissue whereby the medullary canalwas manually cleaned for 30 minutes. The cortical tissue was cut inappropriate rough bone part shapes, a step which lasted anywhere from 30minutes to 3 hours, depending on the donor and bone part shape. Afterrough cutting, the next step involved final machining and part assemblyof the control tissue, taking anywhere from 5 minutes to 5 hoursdepending on the bone part being machined.

Each bone part was then subjected to an ultrasonic bath of a nonionicdetergent consisting of greater than or about 0.1 wt. % TritonX-100 for30 minutes at 34° C., plus or minus 1° C. This ultrasonic bath can alsouse Tween 80 or if desired, another nonionic detergent such asN,N-Dimethyldodecylamino-N-oxide, Octylglucoside, Polyoxyethylene (PEG)alcohols, Polyoxyethylene-p-t-octylphenol, Polyoxyethylene nonylphenol,Polyoxyethylene sorbitol esters, Polyoxy-propylene-polyoxyethyleneesters, p-isoOctylpolyoxy-ethylene-phenol formaldehyde polymer can beused. An ionic detergent will degrade proteins and effectosteoinductivity of the bone graft. The detergent was drained and thebone graft part was subjected to a 5 minute ultrasonic soak of USPpurified water at 34° C., plus or minus 1° C. The water soak was emptiedand repeated, totaling 2 separate soaks to remove the detergent and afinal water soak was instituted comprising a 30 minute continuousultrasonic soak in USP purified water at 34° C., plus or minus 1° C.After the final water soak was emptied, the cortical bone part samplewas ultrasonically cleaned in 3% hydrogen peroxide at 34° C., plus orminus 1° C. for 15 minutes to 2 hours, preferably 1 hour. The hydrogenperoxide concentration can range from 1.5% to 30% depending on the timeand temperature used. After emptying the hydrogen peroxide, the bonepart is again subjected to a 5 minute ultrasonic soak of USP purifiedwater at 34° C., plus or minus 1° C. The soak was emptied and repeatedfor a total of 2 soaks, drained and followed with a 30 minute continuousultrasonic soak in USP purified water at 34° C., plus or minus 1° C.After the purified water soak is emptied, the coral bone sample part wasultrasonically soaked in an alcohol solution SDA-3C (70% EtOH/IPA) at34° C., plus or minus 1° C. for 30 minutes to 2 hours, preferably 1hour, drained and followed by another ultrasonic soak in USP purifiedwater at 34° C., plus or minus 1° C. for 5 minutes. The purified watersoak was emptied and repeated a total of 2 soaks to remove the alcoholand the sample part removed. The sample part was then measured, swabbedand packaged. The package containing the sample undergoes lyophilizationor freeing and then undergoes quality assurance.

Process 2:

In the allograft tissue purification process, cortical bones are takenfrom the normal bone recovery process where they have been frozen andshipped or stored for processing. The bone tissue was thawed in aGentamicin soak (3.1 g Gentamicin in 4000 ml water) for 15 minutes to 2hours. A debridement process was performed, gross cleaning the bonesample with a wire wheel or scalpel for 30 minutes. Condyles were cutfrom the long bones and cortical and cancellous tissue was separated.Processing was begun on the cortical tissue whereby the medullary canalwas manually cleaned for 30 minutes. The cortical tissue was cut inappropriate rough bone part shapes, a step which lasted anywhere from 30minutes to 3 hours, depending on the donor and bone part shape. Afterrough cutting, the next step involved final machining and part assemblyof the control tissue, taking anywhere from 5 minutes to 5 hoursdepending on the bone part being machined.

The vertical bone shaft is subjected to ultrasonic cleaning in 0.1 wt. %TritonX-100 for 1 to 3 hours at 34° C., plus or minus 1° C. The verticalbone shaft is subjected to a pressurized rinse with purified water forup to 1 hour and drained. This cleaning detergent bath of 0.1 wt. %Triton X-100 and rinse is repeated with sonication until clean.

Each bone part was then subjected to an ultrasonic bath of a nonionicdetergent consisting of greater than or about 0.1 wt. % TritonX-100 for30 minutes at 34° C., plus or minus 1° C. This ultrasonic bath can alsouse Tween 80 or if desired, another nonionic detergent such asN,N-Dimethyldodecylamino-N-oxide, Octyglucoside, Polyoxyethylene (PEG)alcohols, Polyoxyethylene-p-t-octylphenol, Polyoxyethylene nonylphenol,Polyoxyethylene sorbitol esters, Polyoxy-propylene-polyoxyethyleneesters, p-isoOctylpolyoxy-ethylene-phenol formaldehyde polymer can beused. An ionic detergent will degrade proteins and effectosteoinductivity of the bone graft. The detergent was drained and thebone graft part was subjected to a 5 minute ultrasonic soak of USPpurified water at 34° C., plus or minus 1° C. The water soak was emptiedand repeated, totaling 2 separate soaks to remove the detergent and afinal water soak was instituted comprising a 30 minute continuousultrasonic soak in USP purified water at 34° C., plus or minus 1° C.After the final water soak was emptied, the cortical bone part samplewas ultrasonically cleaned in 3% hydrogen peroxide at 34° C., plus orminus 1° C. for 15 minutes to 2 hours preferably 1 hour. The hydrogenperoxide concentration can range from 1.5% to 30% depending on the timeand temperature used. After emptying the hydrogen peroxide, the bonepart is again subjected to a 5 minute ultrasonic soak of USP purifiedwater at 34° C., plus or minus 1° C. The soak was emptied and repeatedfor a total of 2 soaks, drained and followed with a 30 minute continuousultrasonic soak in USP purified water at 34° C., plus or minus 1° C.After the purified water soak is emptied, the cortical bone sample partwas ultrasonically soaked in an alcohol solution SDA-3C (70% EtOH/IPA)at 34° C., plus or minus 1° C. for 30 minutes to 2 hours, preferably 1hour, drained and followed by another ultrasonic soak in USP purifiedwater at 34° C., plus or minus 1° C. for 5 mites. The purified watersoak was emptied and repeated a total of 2 soaks to remove the alcoholand the sample part removed. The sample part was then measured, swabbedand packaged. The package containing the sample undergoes lyophilizationor freezing and then undergoes quality assurance.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.However, the invention should not be construed as limited to theparticular embodiments which have been described above. Instead, theembodiments described here should be regarded as illustrative ratherthan restrictive. Variations and changes may be made by others withoutdeparting from the scope of the present inventions defined by thefollowing claims:

1. A method for producing a cleaned sterile bone graft suitable fortransplantation into a human, comprising the steps of: a) sonicating abone graft in a nonionic detergent in an ultrasonic cleaner at atemperature ranging from about 31° C. to about 35° C. and for a timeperiod ranging from 15 minutes to 2 hours effective to produce a cleanedbone graft essentially free from bone marrow; b) sonicating said bonegraft in a purified water in an ultrasonic cleaner at a temperatureranging from about 33° C. to about 37° C. a plurality of times to removethe nonionic detergent; c) sonicating said bone graft in hydrogenperoxide in an ultrasonic cleaner at a temperature ranging from about33° C. to about 37° C. for a time period ranging from 10 minutes toabout 2 hours effective to retain osteoinductivity, d) sonicating saidbone graft in a purified water in an ultrasonic cleaner at a temperatureranging from about 33° C. to about 37° C. a plurality of times to removethe hydrogen peroxide; and e) sonicating said bone graft in an alcoholsolution at a temperature ranging from 33° C. to 37° C. for 30 minutesto 2 hours effective to produce a clean sterile bone graft.
 2. Themethod of claim 1, wherein said time period for the detergent is for atleast 30 minutes and said time period for the purified water is for atleast 30 minutes.
 3. The method of claim 1, wherein said detergent isTriton X-100.
 4. The method of claim 1 wherein said detergent is Tween80.
 5. The method of claim 1 wherein said step of sonicating said bonegraft with hydrogen peroxide is conducted within a time period rangingfrom 15 minutes to 1 hour.
 6. The method of claim 1 wherein said step ofsonicating said bone graft with hydrogen peroxide is conducted within atime period ranging from about 1 hour to about 2 hours.
 7. The method ofclaim 1 wherein said hydrogen peroxide ranges from 1% to 30% inconcentration.
 8. The method of claim 1 wherein said hydrogen peroxideis 3% in concentration.
 9. The method of claim 1, wherein said bonegraft is cortical bone.
 10. The method of claim 1, wherein said bonegraft is cancellous bone.
 11. The method of claim 1, wherein said bonegraft is cortical cancellous bone.
 12. The method of claim 1, whereinsaid bone graft is soft tissue
 13. The method of claim 1 including afurther step (f) sonicating a bone graft in purified water in anultrasonic cleaner at a temperature from about 33° C. to about 37° C. aplurality of times to remove the alcohol from the bone graft.
 14. Themethod of claim 1 wherein said nonionic detergent comprises a memberselected from the group consisting of N,N-Dimethyldodecylamino-N-oxide,Octylglucoside, Polyoxyethylene (PEG) alcohols,Polyoxyethylene-p-t-octylphenol, Polyoxyethylene nonylphenol,Polyoxyethylene sorbitol esters, Polyoxy-propylene-polyoxyethyleneesters, p-isoOctylpolyoxy-ethylene-phenol formaldehyde polymer.
 15. Themethod of claim 1 wherein said alcohol solution comprises an aqueouscombination of ethanol ranging from 40% to 95% and isopropyl alcoholranging between 0% to 10%.
 16. The method of claim 1 wherein viruses arecleared from the bone graft at least two logs.
 17. The method of claim13 wherein the viruses Bovine Viral Diarrhea, Human ImmunodeficiencyVirus and Polio are cleaned from the bone graft at least 10 logs. 18.The method of claim 13 wherein said are Bovine Viral Diarrhea, HumanImmunodeficiency Virus, Pseudorabies, Hepatitis A, Polio and PorcineParvovirus.
 19. The method of claim 13 wherein said hydrogen peroxidestep is effective to clear the viruses Hepatitis C, HumanImmunodeficiency Virus, CMV/Herpes, Hepatitis A Polio/Picornaviridae andHuman Parvovirus B19.
 20. The method of claim 16 wherein the HumanImmunodeficiency Virus is reduced over a quadrillion times (10¹⁵).
 21. Abone graft suitable for transplantation into a human produced by theprocess as claimed in claim 1 wherein bacteria are reduced at least tenlogs.
 22. The method of claim 1 wherein bacteria are reduced from thebone graft at least two logs.
 23. The method of claim 19 wherein thebacteria are Candida albicans, Staphylococcus aureus, Staphylococcusepidermidis, Escherichia coli, Pseudomonas aeruginosa, and Bacillussubtilis are cleaned from the bone graft at least 10 logs.
 24. Themethod of claim 18 wherein said bacteria are Candida albicans,Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli,Pseudomonas aeruginosa, and Bacillus subtilis.
 25. A bone graft suitablefor transplantation into a human produced by the process as claimed inclaim 1 having a bacteria reduction of at least two logs.
 26. The methodof claim 1 wherein the osteoinductivity score of the bone graft afterstep (c) ranges from about 3.50 to about 2.80.
 27. The method of claim 1wherein the osteoinductivity score of the bone graft after the processis over 3.0.
 28. A method for producing a bone graft with a viralclearance of at least two logs suitable for transplantation into ahuman, comprising the steps of: a) sonicating a bone graft with anonionic detergent in an ultrasonic cleaner at a temperature and for atime period effective to produce a cleaned bone graft essentially freefrom bone marrow; b) sonicating said bone graft in purified water in anultrasonic cleaner at a temperature and for a time period effective toremove the detergent; c) sonicating said bone graft in hydrogen peroxidesolution from about 33° C. to about 37° C. for a time period effectiveto produce a bone graft with an osteoinductivity score ranging from 2.0to 3.8; d) sonicating said bone graft in purified water in an ultrasoniccleaner at a temperature and for a time period effective to remove thehydrogen peroxide; e) sonicating said bone graft in an alcohol solutionat a temperature and for a time effective to sterile bacteria at leastfive logs; and f) sonicating said bone graft in purified water in anultrasonic cleaner at a temperature and for a time period effective toremove the alcohol.
 29. The method of claim 28 wherein said nonionicdetergent comprises a member selected from the group consisting of:N,N-Dimethyldodecylamino-N-oxide, Octylglucoside, Polyoxyethylene (PEG)alcohols, Polyoxyethylene-p-t-octylphenol, Polyoxyethylene nonylphenol,Polyoxyethylene sorbitol esters, Polyoxy-propylene-polyoxyethyleneesters, p-isoOctylpolyoxy-ethylene-phenol formaldehyde polymer.
 30. Themethod of claim 28 wherein said alcohol comprises one or more membersselected from the group consisting of ethanol isopropanol, and mixturesthereof.
 31. The method of claim 30 wherein steps (b), (d) and (f) arerepeated a plurality of times.
 32. The method of claim 28 whereinviruses are cleared from the bone graft.
 33. The method of claim 32wherein said clearance is at least two logs.
 34. The method of claim 32wherein said cleared viruses are Bovine Viral Diarrhea, HumanImmunodeficiency Virus, Pseudorabies, Hepatitis A, Polio and PorcineParvovirus.
 35. The method of claim 32 wherein said process is effectiveto clear Hepatitis C, Human Immunodeficiency Virus, CMV/Herpes,Hepatitis A Polio/Picornaviridae and Human Parvovirus B19.
 36. Themethod of claim 35 wherein the Human Immunodeficiency Virus is reducedover a quadrillion times (10¹⁵).
 37. A bone graft suitable fortransplantation into a human produced by the process as claimed in claim28 whereby the bone graft has an osteoinductivity score over 2.0 andviral clearance of at least two logs.
 38. A bone graft suitable fortransplantation into a human produced by the process as claimed in claim28 whereby the bone graft has an osteoinductivity score over 3.0 andviral clearance of at least two logs.
 39. The method of claim 28 whereinsaid alcohol has a 70% to 80% concentration.
 40. The method of claim 39wherein said alcohol comprises one or more solutions selected from thegroup consisting of ethanol, isopropanol and mixtures thereof.
 41. Themethod of claim 28 wherein said hydrogen peroxide ranges from 1.5% to30% by weight.
 42. The method of claim 28 wherein bacteria are reducedfrom the bone graft at two log.
 43. The method of claim 42 wherein thebacteria Candida albicans, Staphylococcus aureus, Staphylococcusepidermidis, Escherichia coli, Pseudomonas aeruginosa, and Bacillussubtilis are cleaned from the bone graft at least ten logs.
 44. Themethod of claim 42 wherein said bacteria are Candida albicans,Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli,Pseudomonas aeruginosa, and Bacillus subtilis.
 45. A bone graft suitablefor transplantation into a human produced by the process as claimed inclaim 28 having a bacteria cleared to at least ten logs.
 46. A methodfor producing a cleaned bone graft suitable for transplantation into ahuman, comprising the steps of: a) sonicating a bone graft in a nonionicdetergent in an ultrasonic cleaner at an energy level ranging frombetween 10-180 W/sq. in. at a temperature from about 33° C. to about 35°C. and for a time period ranging from 15 minute to 1 hour to provide acleaned bone graft essential free from bone marrow; b) sonicating saidbone graft in purified water in an ultrasonic cleaner at an energy levelranging from between 10-180 W/sq. in. at a temperature from about 33° C.to about 35° C. and for a time period effective to remove the detergent;c) sonicating said bone graft in a solution of hydrogen peroxide rangingin strength from about 1.5% to about 30% at an energy level ranging frombetween 10-180 W/sq in. at a temperature above 33° C. to about 35° C.for 15 minutes to 3 hours to produce a bone graft having anosteoinductivity score of at least 2.0; d) sonicating said bone graft inpurified water in an ultrasonic cleaner at an energy level ranging from10-180 W/sq. in. at a temperature ranging from about 33° C. to about 35°C. and for a time period effective to remove the hydrogen peroxide; e)sonicating said bone graft in an alcohol in an ultrasonic cleaner at anenergy level ranging from 10-180 W/sq. in. at a temperature ranging fromabout 33° C. to about 35° C. for a time effective to reduce organisms atleast two logs; and f) sonicating said bone graft in purified water inan ultrasonic cleaner at an energy level ranging from 10-180 W/sq. in.at a temperature ranging from about 33° C. to about 35° C. for a timeperiod effective to remove the alcohol.
 47. A method for producing acleaned pre-shaped bone g suitable for transplantation into a human,comprising the steps of: a) sonicating a bone graft with a nonionicdetergent in an ultrasonic cleaner with a pressurize rinse at atemperature and for a time period effective to produce a substantiallycleaned bone graft; b) cutting the bone graft into an appropriate shapeor shapes; c) sonicating a bone graft in a nonionic detergent in anultrasonic cleaner at a temperature and for a time period ranging from15 minute to 1 hour to produce a cleaned bone graft essentially freefrom bone marrow; d) sonicating said bone graft in purified water in anultrasonic cleaner at a temperature and for a time period effective toremove the detergent; e) sonicating said bone graft in a solution ofhydrogen peroxide ranging from about 1.5% to about 30% at a temperatureand for a time period effective to produce a bone graft to reduceviruses at least two logs having an osteoinductivity score of at least2.0; f) sonicating said bone graft in purified water in an ultrasoniccleaner at a temperature and for a time period effective to remove thehydrogen peroxide; and g) sonicating said bone graft with an alcohol ata temperature and for a time effective to reduce bacteria and clearviruses at least two logs.
 48. The method of claim 47 wherein saidalcohol comprises one or more members selected from the group consistingof ethanol, isopropanol, and mixtures thereof.